Post-translational modification of proteins has long been regarded as central to the regulation of cellular processes. Modification of proteins may affect their biological activity, or may affect their binding affinities for other cellular components, or may mark a protein for degradation, or have many other effect(s) on the protein. However, monitoring the post-translational modification state of proteins has been fraught with difficulties.
Even monitoring the abundance (for example the presence or absence) of a protein has been a laborious task, requiring extraction of proteins, followed by some kind of immuuno-assay such as ELISA testing, Western-blotting, slot- or dot-blotting, immunoprecipitation or an enzyme assay for the activity of the protein. These methods are themselves time-consuming, and demand that the necessary reagents have already been prepared, such as antibodies which react with the protein of interest, or an assay which is able to read out the enzymatic activity of the polypeptide of interest. Even the preparation of these essential reagents requires significant investment of resources, and not all polypeptides have known enzymatic activities which can be readily assayed.
Monitoring the post-translational modification of various proteins has been accomplished in the past by extension of those techniques outlined above. For example, monitoring of ubiquitination of proteins has been carried out by extracting proteins from the sample to be tested, immunoprecipitating the protein of interest, Western blotting the immunoprecipitated protein, and then developing the Western blot using an anti-ubiquitin antibody. Clearly, this is a complicated procedure. In addition, there are many different isoforms of ubiquitin, and so repetition of the assay with antibodies against all the various forms of ubiquitin can increase the effort involved manifold.
Other types of modification which have in the past been monitored include phosphorylation. This may often be monitored in a manner analogous to the monitoring of ubiquitin, using phosphorylation-specific antibodies to immunoprecipitate and/or immunoblot only phosphorylated form(s) of the protein. However, the raising of phospho-specific antibodies is an expensive proposition, requiring careful screening of large numbers of different antisera. Moreover, the production of such reagents, even if attempted, is not always possible. For example, there is simply no reliable antibody available for the detection of phospho-serine or phospho-threonine. Currently, the only way of detecting these phosphorylation events is either to embark upon a programme to raise context-specific anti-phospho-antisera, which is not always feasible, or to use radioactive labelling techniques in conjunction with standard immunoprecipitation, assuming that immunoprecipitating antibodies are available to the protein which is to be tested. Clearly, it is simply not possible to use radioactive labelling when dealing with human patients.
Furthermore, parallel analysis of more than one of these parameters at a time is not possible. Each of the desired data must be tested individually, requiring larger samples of the starting material, and greater amounts of technicians' time. Building up a picture of the many variables which may be present in a disease state may require dozens of discrete tests, and concomitant expenditure of resources.
The present invention seeks to overcome such difficulties.